This project studies cellular and physiological aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration ([Ca2+]i) that is strongly modulated by the transport activity of intracellular calcium storage organelles. This transport activity plays an important role in spatio-temporally shaping the signals that regulate processes like gene expression and synaptic plasticity. We (and others) had earlier shown that stimulus-induced increases in [Ca2+]i in a variety of neurons induce large, reversible elevations in the concentration of calcium within mitochondria, which in turn has important effects on physiological and pathophysiological processes. This year we continued to explore the consequences of mitochondrial Ca2+ uptake, showing that in hippocampal pyramidal neurons this activity at least partly underlies the phenomenon of ischemic preconditioning. We have also further characterized the steps in the ERK1/2 signal transduction cascade that are regulated by mitochondrial calcium. It was previously shown that NMDA overstimulation that leads to excitotoxic delayed cell death (DCD) is inevitably associated with strong mitochondrial calcium accumulation. This mitochondrial activity is spatially heterogeneous, which is important because cell vulnerability is correlated with the aggregate size of the mitochondrial Ca load and the number of Ca-overloaded, damaged mitochondria in a given neuron. Thus, the number and location of damaged mitochondria are thought to determine vulnerability to excessive NMDA. These observations have led us to explore the hypothesis that mitochondria also mediate the neuroprotective effects of ischemic preconditioning (PC), a poorly understood phenomenon whereby neurons become resistant to a normally lethal insult after pretreatment with a similar but milder, non-lethal challenge. The results from several recognized preconditioning protocols showed that at the cellular level these treatments recapitulate the effects of NMDA by inducing the reversible redistribution of intracellular Ca and the transient depolarization and Ca loading of mitochondria. Such effects were fully reversible and did not lead to DCD. Among the protocols tested were: 1) so-called chemical ischemia (CI-PC), that is, exposure to 2 mM cyanide in glucose-free medium for 30 min 24h before ?lethal? NMDA; and 2) various repetitive (two to five) treatments with normally lethal concentrations of NMDA but for shorter times, spaced 48 h apart with the last 24h before lethal insult (NMDA-PC). Exposure to lethal NMDA 24h after CI-PC reduced NMDA-induced cell death by >30%, whereas NMDA-PC was even more effective, reducing cell death as much as 90%. In both cases, PC neuroprotection was paralleled by reduced mitochondrial injury after excitotoxic NMDA exposure. However, NMDA-PC (multiple exposures) also substantially reduced Ca2+ entry, while CI-PC did not. These results support the working hypothesis that PC exerts its protective effect by increasing mitochondrial tolerance for large Ca loads, thereby attenuating mitochondrial dysfunction. More effective PC protocols appear to recruit additional, additive protective mechanisms that are yet to be defined. In hippocampal neurons large [Ca2+]i increases activate several important kinases, e.g., Ca/calmodulin-dependent kinases (CaMKs), which is important because these enzymes regulate pathways central to gene expression and neuronal plasticity. Our laboratory previously reported that mitochondrial calcium accumulation mediated by strong Ca2+ entry leads to an increase in the production of superoxide radicals (O2-), which up-regulates the activity of several kinases, including PKA, PKC, CaMKII, nuclear CaMKIV (and therefore the transcription factor CREB), and ERK 1/2. Up-regulation of all but PKC occurs by a common mechanism, namely, stabilization of the active, phosphorylated form of the kinase by O2- inhibition of the deactivating serine/threonine protein phosphatases. In all these cases, mitochondria are the main source of active O2-, but ERK 1/2 is different in that NADPH oxidase-derived O2- also plays a significant role. This year we have continued to expand earlier studies showing that the more complex regulation of the Ras/Raf/MEK/ERK cascade arises because key steps in this cascade depend on both S/T protein phosphatase and protein tyrosine phosphatase activities, which are differentially regulated by O2- from different sources. Thus, NADPH oxidase generates a brief spike of O2- that promotes the initial activation of Ras, whereas mitochondria produce sustained O2- elevations that affect later phosphatase-dependent steps. There are several parallel pathways in the ERK cascade; the best characterized of these -- (Ras)/Raf-1/MEK/ERK and Rap-1/B-Raf/MEK/ERK -- converge at MEK and are activated by different stimuli. Since stimulus patterns are generally important for specifying the targets of Ca2+ signals, we are examining how these two branches of the ERK cascade respond to different trains of high-frequency stimuli. Quantitative immunocytochemistry showed that at 1 min after a 100Hz/18s stimulus both Raf-1 and B-Raf pathways were activated. In contrast, three episodes of such stimulation at 5-min intervals selectively activated B-Raf, which was found to be dependent on PKA and Pyk-2, but not Src. This indicates the long-term induction of a PKA-dependent, Src-independent Rap-1/B-Raf pathway, and probably the suppression of the Raf-1 pathway. Thus, Raf vs. Rap appears to be yet another checkpoint at which O2- modulation of kinase signaling can fine-tune the route and timing of the ERK cascade.